Published April 22, 2021 | Published
Journal Article Open

Proteomics of protein trafficking by in vivo tissue-specific labeling

  • 1. ROR icon Harvard University
  • 2. ROR icon Scripps Research Institute
  • 3. ROR icon University of Southern California
  • 4. ROR icon China Agricultural University
  • 5. ROR icon Broad Institute
  • 6. ROR icon Yale University
  • 7. ROR icon CZ Biohub
  • 8. ROR icon Beth Israel Deaconess Medical Center
  • 9. ROR icon Stanford University
  • 10. ROR icon Howard Hughes Medical Institute

Abstract

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

Copyright and License

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Acknowledgement

I.A.D. acknowledges generous financial support from NSERC PGS-M and PGS-D, Brigham and Women’s Hospital Osher Pre-doctoral fellowship, and Harvard Medical School (HMS) Department of Cell Biology Innovative Grant Program (IGP). We thank Aldina Mesic and the Microscopy Resources on the North Quad (MicRoN) core at HMS for excellent technical assistance. We thank the Janelia Gene Targeting and Transgenics Facility (Director: Caiying Guo) for the mESC generation. We thank Seung Kim, Andreas Brech, Akhila Rajan, TRiP at HMS, VDRC, Bloomington Drosophila Resource Center, and Developmental Studies Hybridoma Bank for stocks and antibodies. N.P. is an investigator of the Howard Hughes Medical Institute (HHMI). We acknowledge the National Institutes of Health (NIH) grants 5P01CA120964 (J.M.A.) and 5P30CA006516 (J.M.A.). This work was supported by an HCIA grant from HHMI, NIGMS P41 GM132087 to N.P., and the NIH Transformative R01 grant 5R01DK121409 to A.P.M., A.Y.T., S.A.C., and N.P.

Funding

We acknowledge the National Institutes of Health (NIH) grants 5P01CA120964 (J.M.A.) and 5P30CA006516 (J.M.A.). This work was supported by an HCIA grant from HHMI, NIGMS P41 GM132087 to N.P., and the NIH Transformative R01 grant 5R01DK121409 to A.P.M., A.Y.T., S.A.C., and N.P.

Contributions

I.A.D. conceived the initial Drosophila method and study, and I.A.D., A.P.M., and N.P. conceived the extension of the approach to mammals. I.A.D., A.S.M., A.P.M., and N.P. designed and supervised experiments. I.A.D., D.W., A.S.M., R.Y., J.A.M., J.G., N.D.U., L.M., D.K.C., D.R., T.S., R.Z., A.T., and J.M.A. performed experiments. I.A.D., D.W., A.S.M., R.Y., J.M., Y.H., N.D.U., L.M., D.K.C., T.S., J.M.A., S.A.C., A.P.M., and N.P. analyzed data. T.B. and A.Y.T. generated BirA*G3, and J.A.B. cloned it into Drosophila. I.A.D, A.S.M., and N.P. wrote the manuscript with input from all authors.

Data Availability

The original mass spectra for all experiments, and the protein sequence databases used for searches have been deposited in the public proteomics repository MassIVE (https://massive.ucsd.edu) and are accessible at https://doi.org/10.25345/C5NB8W with the accession number MSV000086664 (mouse and fly BirA* data sets), and at https://doi.org/10.25345/C5XN4Z with the accession number MSV000086291 (fly hemolymph data sets). The following publicly available data sets/databases were used: UniProt database (https://www.uniprot.org/Drosophila (DROME; https://www.uniprot.org/proteomes/UP000000803), mouse (https://www.uniprot.org/proteomes/UP000000589), and human (https://www.uniprot.org/proteomes/UP000005640)), DIOPT (https://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl), GLAD (https://www.flyrnai.org/tools/glad/web/), Signaling Receptome (http://www.receptome.org/), human plasma proteome data sets (http://www.peptideatlas.org/repository/repository_public_Hs_Plasma2.php (PeptideAtlas) and Supplementary Table 1 in ref. 61), SignalP database (http://www.cbs.dtu.dk/services/SignalP/ and http://www.cbs.dtu.dk/services/SignalP-4.1/), Drosophila mitochondrial proteome (Supplementary Data 1 in ref. 52), TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP/), FlyAtlas microarray (http://flyatlas.org/atlas.cgi), Drosophila RNAseq (http://www.modencode.org/celniker/), PAXdb (https://pax-db.org/), NCBI Gene (https://www.ncbi.nlm.nih.gov/gene/), FlyBase (https://flybase.org/), mammalian adipocyte secretomes (Tables S1–S5 in ref. 7, Supplementary Data Table C in ref. 70, Supplementary Data Table in ref. 71, Supplementary Table S1 in ref. 72, Supplementary Table—Secretome in ref. 73, Supplementary Table S1 in ref. 74, Data File S1 in ref. 75, Table 2 and Supplementary Table 1 in ref. 77, Supplementary Table 2A in ref. 78), mammalian myocyte secretomes (Table 1 in ref. 79, Supplementary Table S1 in ref. 80, Supplementary Table 1 in ref. 81, Supplementary Table 1 in ref. 82, Table 2 in ref. 83, Supplementary Table S1 in ref. 84, Table S5 in ref. 85, Supplementary Table S1 in ref. 86). The main text and supplementary information shows all of the data collected as part of this work. The corresponding authors will provide original data on reasonable request. Source data are provided with this paper.

Supplemental Material

  • Description of additional supplementary files:
  • Supplementary Data 1: BirA*G3-ER mass spectrometry data (in separate Excel attachment)

  • Supplementary Data 2: Total hemolymph mass spectrometry data (in separate Excel attachment)

  • Supplementary Data 3: BirA*R118G-ER mass spectrometry data (in separate Excel attachment)

  • Supplementary Data 4: Teratoma BirA*G3-ER mass spectrometry data (in separate Excel attachment). Two-sided two-sample t-test unadjusted p-values shown.

  • Supplementary Data 5: Serum BirA*G3-ER mass spectrometry data (in separate Excel attachment). Two-sided two-sample t-test unadjusted p-values shown.

  • Supplementary Data 6: Examples of interesting proteins identified in the teratoma-derived serum proteomics dataset (in separate Excel attachment)

  • Supplementary Movie 1: : LPP-Gal4>control (attp) (vial 1; left) versus LPP-Gal4>CG2145-i-1 (vial2; right) climbing-ability assay at 3 weeks old and 29°C. Flies were tapped to the bottom of the vial at the beginning of the assay and then allowed to climb up the vial.

  • Supplementary Movie 2: LPP-Gal4>control (w-i) (vial 1; left) versus LPP-Gal4>CG4332-i-1 (vial 3; right) climbing-ability assay at 5 weeks old and 27°C. Flies were tapped to the bottom of the vial at the beginning of the assay and then allowed to climb up the vial.

  • Supplementary Movie 3: : LPP-Gal4>control (w-i) (vial 1; left) versus LPP-Gal4>CG31326-i-3 (vial 2; right) climbing-ability assay at 5 weeks old and 27°C. Flies were tapped to the bottom of the vial at the beginning of the assay and then allowed to climb up the vial.

 

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Additional details

Created:
November 16, 2024
Modified:
November 16, 2024